Consequences of Combining mtDNAandnucDNAData for Phylogenetic TreesLeila Bahmani Kazerooni & John Malloy
What is a Phylogenetic Tree? Leaves• Evolutionary tree (Species) Node Human Root Chimp Gorilla Orangutan Branch (Edge)• Visually defines species relationships Deep divergences (e.g., The Tree of Life) Recent divergences (e.g., species in a genus)
What is a Phylogenetic Tree? Leaves• Evolutionary tree (Species) Node Human Root Chimp Gorilla Orangutan Branch (Edge) • Information required to build a phylogeny ranges from morphological data to purely genomic data, including mitochondrial DNA (mtDNA) and nuclear DNA (nucDNA)
Molecular Phylogenies-mtDNA• Up until now, phylogenetic trees of closely related animals have mostly been based on one region of the genome - mitochondrial DNA (mtDNA) High mutation rate (due to poor DNA repair mechanism) Passed down through the maternal lineage
Molecular Phylogenies-nucDNA• Nuclear DNA (nucDNA) has recently become a valuable tool alongside mitochondrial DNA Slower mutation rate Inherited from both parents More versatile - contains both sex-linked and autosomal genes (allows for increased validation)
Concatenation=(mtDNA+nucDNA)• Both mtDNA and nucDNA have unique advantages and disadvantages• A method used to give both regions of the genome an opportunity to resolve the branches on the tree is CONCATENATION Concatenation: Combining a mitochondrial gene with multiple nuclear genes to form one “BIG gene”• This frequently used method may be flawed due to potential gene flow between species.• We have used the “Northern Oriole” group as a model group to test concatenation
“Northern Oriole” Group: Gene Flow BetweenSpecies HYBRID ZONE EASTERN WESTERN WESTERN Baltimore Bullock’s Bullock’s MEXICAN Black-backed MEXICAN Black-backed •These three species illustrate one of the main challenges in determining relationshi among recently diverged species.
Constructing the Phylogeny-MATERIALS• Samples from 32 species of the genus Icterus were used in the analyses• The mtDNA used belong to the cytochrome b and ND2 regions and had been previously isolated and sequenced by the Omland lab 1.• The nucDNA had also been isolated and sequenced by the Omland lab. Six z-linked introns: ADAM-5, ALDO-5, BRM-15, CHD-18, MUSK-3, SLC- 92 Six autosomal introns: β-ACT2, αENO8, FGB4, GAPDH11, RDP2, TGFβ5 3
Constructing the Phylogeny-TOOLS• The formatting and concatenation of the data files were done using the modular file editor, Mesquite v2.75 4.• Files from Mesquite were exported in the Nexus file format and analyzed with MrBayes. MrBayes is a tree building program that uses Bayesian inference and Markov chain Monte Carlo methods to infer phylogenetic trees 5.• Output trace files from MrBayes were assessed using Tracer, to ensure the convergence of each run 6.• MrBayes outputs were converted to graphical trees using FigTree, a tree viewing program7.
Tested nucDNA against mtDNA – Deep divergence agreed: CLADE BnDNA (6 Z-introns combined) mtDNA Jacobsen, Friedman &Omland 2010
Tested nucDNA against mtDNA – Deep divergence agreed: CLADE CnDNA (6 Z-introns combined) mtDNA Jacobsen, Friedman &Omland 2010
Tested nucDNA against mtDNA – Deep divergence agreed BUT one recent divergence did not nDNA (6 Z-introns combined) mtDNA Bullock’s Black Backed Bullock’s Black Backed Baltimore Baltimore Jacobsen, Friedman &Omland 2010
Summary of the Conflicting Nodes in Clade C combined nucDNA tree VS mtDNA gene tree nucDNA Baltimore mtDNA Black-backed Bullock’s•Previous analyses done by the Omland lab have shown strong support that themtDNA tree is misleading, while there is strong support for the nucDNA tree1.•All analyses of nuclear DNA – z chromosome alone, autosomal chromosomesalone, and different analysis methods – all show mtDNA is the problematic outlier1,2.
mtDNA and the problem with mindless CONCATENATION• Why might mtDNA and nucDNA disagree for the Northern Oriole Group? Gene flow – mtDNA may have crossed between species during hybridization• What if we had mindlessly tried to resolve the conflict between mtDNA and nucDNA by simply combining both types of DNA into one large data matrix (Concatenation)? Give both nucDNA and mtDNA an "equal" opportunity to resolve the branches in the tree (very common approach in molecular systematics)
Concatenate 12nuc+1mtmt Black-backed nuc Black-backed Baltimore Bullock’s Bullock’s Baltimore Black-backed12nuc+mt Baltimore • We combined 1 mitochondrial gene and 12 nuclear genes (6 sex-linked and 6 autosomal) •The mitochondrial signal overwhelmed the nuclear signal. Bullock’s
Concatenate 24nuc+1mtmt Black-backed nuc Black-backed Baltimore Bullock’s Bullock’s Baltimore24nuc+mt Black-backed •To further test the influence of Baltimore mtDNA on the branches, we combined 24 nuclear genes and 1 mitochondrial gene, thereby having doubled the nuclear signal. •The mitochondrial signal still Bullock’s overwhelmed the nuclear signal.
Concatenate 48nuc+1mtmt Black-backed nuc Black-backed Baltimore Bullock’s Bullock’s Baltimore48nuc+mt Black-backed •Continuing the titration we Baltimore combined 48 nuclear genes and 1 mitochondrial gene, thus quadrupling the nuclear signal. •The mitochondrial signal continued to overwhelm the nuclear signal. Bullock’s
Concatenate 72nuc+1mtmt Black-backed nuc Black-backed Baltimore Bullock’s Bullock’s Baltimore72nuc+mt Black-backed •We then combined 72 Bullock’s nuclear genes and one mitochondrial gene having Baltimore multiplied the original nuclear signal by six. •Only at that point did the nuclear signal overwhelm the mitochondrial signal.
Titrate mtDNA with nucDNA As the nucDNA was artificially doubled and quadrupled the mtDNA signal continued to overwhelm the nucDNA signal mt 12nuc+mt 72nuc+m NODE 24nuc+mt 48nuc+mt nuc t BLACK- ___ ___ BACKED, Y Y Y YBALTIMORE BLACK- __ ___ ___ ___ BACKED, Y YBULLOCK’S Once the nucDNA was artificially multiplied by six the nucDNA signal revealed itself.
Conclusion: mtDNA Potentially Very Misleading• Although the number of nuclear nucleotides overwhelmed mitochondrial nucleotides, the mitochondrial signal remained dominant. mtDNA is much more variable, due to its high mutation rate• Simple concatenation, 12nuc+1mt, proceeded to result in the misleading mtDNA signal.• Only when the nucDNA signal was multiplied by six was the signal strong enough to result in the well supported nuclear tree.• These results consistently demonstrate the potential weaknesses of using concatenation as a technique to build molecular phylogenetic trees.
AcknowledgmentsThanks to Dr. Kevin Omland, Dr. Matthias Gobbert, and Dr. FrodeJacobsen for the contributions and support for this project.This work was funded, in part, by the UBM program at UMBC, aninterdisciplinary biology and math training grant funded by the NationalScience Foundation. Work Cited1 Frode Jacobsen, Kevin E. Omland, Species tree inference in a recent radiation of orioles (Genus Icterus):Multiple markers and methods reveal cytonuclear discordance in the northern oriole group, MolecularPhylogenetics and Evolution, Volume 61, Issue 2, November 2011, Pages 460-469.2 Jacobsen, F., Friedman, N. R. and Omland, K. E. 2010. Congruence between nuclear and mitochondrialDNA: combination of multiple nuclear introns resolves a well-supported phylogeny of New World orioles(Icterus). Molecular Phylogenetics and Evolution 56:419-427.3 Jacobsen, F. and Omland, K. E. 2012. Extensive introgressive hybridization within the northern oriole group(Genus Icterus) revealed by three-species isolation with migration analysis. Ecology and Evolution 2:2413-2429.4 Maddison, W. P. and D.R. Maddison. 2011. Mesquite: a modular system for evolutionary analysis. Version2.75 http://mesquiteproject.org5Huelsenbeck, J. P. and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754-755.6 Rambaut A, Drummond AJ (2007) Tracer v1.4.7 http://tree.bio.ed.ac.uk/software/figtree/